The broad objective of the applicant is to increase our understanding of the function of the vascular endothelium in normal and pathophysiological regulation of the cardiovascular system. The goal of this project is to define the biosynthetic processing for a newly discovered cardiovascular regulatory peptide, endothelin. Work by others and from our own laboratory clearly demonstrates that endothelin is a peptide with powerful actions on a wide variety of physiological functions which impact the cardiovascular system. Little is known about the biosynthetic processing and regulation of this important new factor. Experiments described in this application are directed at isolating and characterizing the endothelin converting enzyme using conventional biochemical methodology. Molecular biology techniques will be used to address substrate requirements of the converting enzyme and ultimately to isolate, clone and express the enzyme itself in quantity. Purified enzyme will be used for extensive biochemical and structural characterization of the enzyme. Pharmacology techniques will be used to verify findings in cultured cell systems and to use the information derived from biochemical and molecular biology investigations to begin to elucidate the pathophysiological significance of endothelin. Specifically, the aims are to demonstrate that a specific endothelin converting enzyme does exist, to develop a convenient assay for the measurement of endothelin converting enzyme activity, to determine the class of proteases the enzyme belongs to and to determine the cellular location of the enzyme, to determine the substrate requirements of the enzyme and ultimately to clone, express and purity endothelin converting enzyme. Aims also address the regulation of endothelin converting enzyme as well as regulation of release of endothelin and the physiological implications of interfering with endothelin synthesis and release. This studies outlined in this proposal have important application to both a greater understanding of the role of the endothelium in cardiovascular disease processes involving vasoconstrictor mechanisms and for future development of therapeutic agents to manipulate the system for the treatment of associated clinical disease. Disease states which endothelin is likely to be involved are ischemic injury, such as myocardial infarction, acute renal failure and stroke. Endothelin may also be important in hypertension and atherosclerosis.